title: Periodontal fibroblasts modulate proliferation and osteogenic differentiation of embryonic stem cells through production of fibroblast growth factors.
abstract: BACKGROUND
  Periodontal ligament fibroblasts (PLFs) maintain homeostasis of periodontal ligaments by producing paracrine factors that affect various functions of stem-like cells. It is hypothesized that PLFs induce proliferation and differentiation of stem cells more effectively than gingival fibroblasts (GFs) and skin fibroblasts (SFs).
  
  
  METHODS
  PLFs and GFs were isolated from extracted teeth and cultured in the presence and absence of osteogenesis-inducing factors. Mouse embryonic stem (mES) cells and SFs were purchased commercially. mES cells were incubated with culture supernatants of these fibroblasts or cocultured directly with the cells. Proliferation and mineralization in mES cells were determined at various times of incubation. Immunostaining and polymerase chain reaction were performed. The activity of mitogen-activated protein kinase and alkaline phosphatase (ALP) was also measured.
  
  
  RESULTS
  In cocultures, PLFs stimulated proliferation of mES cells more effectively than GFs or SFs. Similarly, the addition of culture supernatant of PLFs induced the most prominent proliferation of mES cells, and this was significantly inhibited by treatment with antibody against fibroblast growth factor (FGF)4 or the c-Jun N-terminal kinase inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one). Supplementation with culture supernatant from the fibroblasts induced osteogenic differentiation of mES cells in the order PLFs > GFs > SFs. These activities of PLFs were related to their potential to produce osteogenic markers, such as ALP and runt-related transcription factor-2 (Runx2), and to secrete FGF7. Pretreatment of mES cells with the extracellular signal-regulated kinase inhibitor PD98059 [2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one] or SP600125 clearly attenuated mineralization induced by culture supernatant of PLF with attendant decreases in mRNA levels of Runx2, bone sialoprotein, osteocalcin, and osteopontin.
  
  
  CONCLUSIONS
  PLFs regulate the proliferation and osteogenic differentiation of mES cells more strongly than GFs and SFs via the secretion of FGF through a mechanism that involves mitogen-activated protein kinase-mediated signaling.
authors.affiliation: Institute of Oral Biosciences and School of Dentistry, Chonbuk National University, Jeonju, South Korea.
authors.forename: Sung-Ho
authors.initials: SH
authors.lastname: Kook
authors.forename: Young-Mi
authors.initials: YM
authors.lastname: Jeon
authors.forename: Song-Soo
authors.initials: SS
authors.lastname: Park
authors.forename: Jeong-Chae
authors.initials: JC
authors.lastname: Lee
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation; D018920:Coculture Techniques; D050659:Core Binding Factor Alpha 1 Subunit / Q000032:analysis; D017077:Culture Media, Conditioned; D053595:Embryonic Stem Cells / Q000502:physiology*; D051519:Fibroblast Growth Factor 4 / Q000037:antagonists & inhibitors; D051523:Fibroblast Growth Factor 7 / Q000032:analysis; D005346:Fibroblast Growth Factors / Q000032:analysis / Q000502:physiology*; D005347:Fibroblasts / Q000502:physiology*; D005419:Flavonoids / Q000494:pharmacology; D005881:Gingiva / Q000166:cytology; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D020935:MAP Kinase Signaling System / Q000502:physiology; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis; D015675:Osteocalcin / Q000032:analysis; D010012:Osteogenesis / Q000502:physiology*; D053495:Osteopontin / Q000032:analysis; D010513:Periodontal Ligament / Q000166:cytology*; D012867:Skin / Q000166:cytology
chemical_list: D000873:Anthracenes; D050659:Core Binding Factor Alpha 1 Subunit; D017077:Culture Media, Conditioned; C496446:Fgf4 protein, mouse; C496454:Fgf7 protein, mouse; D051519:Fibroblast Growth Factor 4; D005419:Flavonoids; C493716:Runx2 protein, mouse; C506896:Spp1 protein, mouse; D015675:Osteocalcin; D053495:Osteopontin; D051523:Fibroblast Growth Factor 7; C432165:pyrazolanthrone; D005346:Fibroblast Growth Factors; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D000469:Alkaline Phosphatase; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one